Preparation of biological monolayers for producing high-resolution scanning electron micrographs

Scanning electron microscopy (SEM) provides a technical platform for nanoscopic mapping of biological structures. Correct preparation of SEM samples can provide an unprecedented understanding of the nexus between cellular morphology and topography. This comparative study critically examines two coating methods for preparing biological samples for scanning electron microscopy, while also providing novel advice on how to prepare in vitro epithelial or endothelial samples for high-resolution scanning-electron microscopy (HR-SEM). Two obstacles often confront the biologist when investigating cellular structures grown under tissue culture conditions, namely., how to prepare and present the biological samples to the HR-SEM microscope without affecting topographical membrane and cellular structural alterations. Firstly, our use of the Millicell cellulose inserts on which to grow our cellular samples in preparation for HR-SEM is both novel and advantageous to comparing the permeability function of cells to their morphological function. Secondly, biological material is often non-conducting, thermally sensitive and fragile and, therefore, needs to be fixed correctly and coated with thin conducting metal to ensure high-resolution detail of samples. Immortalized mouse brain endothelial cells (bEnd5) was used as a basis for describing the preferences in the use of the protocol. We compare two biological sample coating modalities for the visualizing and analysis of texturized, topographical, membranous ultrastructures of brain endothelial cell (BEC) confluent monolayers, namely, carbon and gold:palladium (Au:Pd) sputter coating in preparation for HR-SEM. BEC monolayers sputter-coated with these two modalities produced three-dimensional micrographs which have distinctly different topographical detail from which the nanostructural cellular data can be examined. The two coating methods display differences in the amount of nanoscopic detail that could be resolved in the nanosized membrane cytoarchitecture of BEC monolayers. The micrographical data clearly showed that Au:Pd sputter-coated samples generate descript imagery, providing useful information for profiling membrane nanostructures compared to carbon-coated samples. The recommendations regarding the contrast in two modalities would provide the necessary guidance to biological microscopists in preparing tissue culture samples for HR-SEM.

Scanning electron microscopy (SEM) provides a technical platform for nanoscopic mapping of biological structures. Correct preparation of SEM samples can provide an unprecedented understanding of the nexus between cellular morphology and topography. This comparative study critically examines two coating methods for preparing biological samples for scanning electron microscopy, while also providing novel advice on how to prepare in vitro epithelial or endothelial samples for high-resolution scanning-electron microscopy (HR-SEM). Two obstacles often confront the biologist when investigating cellular structures grown under tissue culture conditions, viz., how to prepare and present the biological samples to the HR-SEM microscope without affecting topographical membrane and cellular structural alterations. Firstly, our use of the Millicell cellulose inserts on which to grow our cellular samples in preparation for HR-SEM is both novel and advantageous to comparing the permeability function of cells to their morphological function. Secondly, biological material is often non-conducting, thermally sensitive and fragile and, therefore, needs to be fixed correctly and coated with thin conducting metal to ensure high-resolution detail of samples. Immortalized mouse brain endothelial cells (bEnd5) was used as a basis for describing the preferences in the use of the protocol. We compare two biological sample coating modalities for the visualizing and analysis of texturized, topographical, membranous ultrastructures of brain endothelial cell (BEC) confluent monolayers, namely, carbon and gold:palladium (Au:Pd) sputter coating in preparation for HR-SEM. BEC monolayers sputter-coated with these two modalities produced three-dimensional micrographs which have distinctly different topographical detail from which the nanostructural cellular data can be examined. The two coating methods display differences in the amount of nanoscopic detail that could be resolved in the nanosized membrane cytoarchitecture of BEC monolayers. The micrographical data clearly showed that Au:Pd sputter-coated samples generate descript imagery, providing useful information for profiling membrane nanostructures compared to carbon-coated samples. The recommendations regarding the contrast in two modalities would provide the necessary guidance to biological microscopists in preparing tissue culture samples for HR-SEM.

Materials and methods Materials and methods
High-resolution scanning electron microscopy High-resolution scanning electron microscopy When using HR-SEM, the signal generating the image occurs as a result of the interaction of the primary electron beam with the biological specimen. Briefly, upon interaction, the primary beam electrons induce ionization of the sample's atoms and the subsequent emission of secondary electrons (SE) from the top-most region of the specimen. The surface-emitted SEs are detected by a scintillator-based Everhart-Thornley detector (ETD), also known as secondary or SE detector. Modern SEMs, however, are more frequently manufactured with in-lens SE detectors, which have the ability to detect fine structures that are invisible to traditional ETDs (Griffin, 2011).
For biological materials, which are predominantly hydrocarbons, a low primary beam energy is desirable to minimize the interaction volume depth in accordance with equation (1). A small volume allows the operator to study finer specimen surface detail while simultaneously minimizing sample charging caused by secondary electron build-up on the surface. One drawback, however, is a low signal-to-noise ratio caused by the reduced secondary electron emission. This can, however, be solved by coating the sample surface with a thin layer of conducting material such as gold or graphite. The nexus of structural biology is achieving three-dimensionality and investigating the correlation between the morphological framework and its molecular underpinnings. The macromolecular structure is concomitant with its physiology as the shape of any given structure determines its function. Studying both the nanostructural and/or molecular machinery that governs the phenotypic evolution of a brain endothelial cell (BEC) unifies our understanding of blood-brain barrier (BBB) construction. The volume created by electron beam when incident on the specimen surface is called the interaction volume and is dependent on the following important parameters: (i) the primary electron beam energy (E 0 ), (ii) the average atomic number (Z), (iii) density (ρ) and (iv) average atomic mass (A) of the specimen under investigation (El Azzouzi et al., 2017). A semi-empirical model of the interaction volume depth, R p is given by Kanaya and Okayama, (1972). (1) In this study, specimens were analyzed using a Zeiss Auriga high-resolution field-emission gun SEM (FEG-SEM), operated at an electron beam energy of 5 keV, a nominal working distance of 5mm and using an in-lens SE detector for high-resolution imaging.
bEnd5 Cells were seeded at 5x10 5 cells/insert on a Millicell, mixed cellulose esters insert membrane (Millipore/Merck, Cat no. PIHA01250, Germany). After exposure of the cell monolayers to the standard culture medium (i.e. supplemented DMEM:F12), at respective time intervals (24-48h), the bEnd5 cells were fixed with 2.5% glutaraldehyde made by adding 10 ml of a 25% solutions of glutaraldehyde (Fluka/ Sigma, Cat no. 49626, Switzerland) in 90 ml of 1X phosphate-buffered saline (PBS) solution (Life Technologies, Cat. no.20012019 , South Africa). Buffers and fixatives used in culture were maintained at pH 7.2 and an osmolality which equaled that of blood plasma (280-300 mOs/kg) utilizing a Vapor pressure osmometer (VAPRO) (Wescor, South Africa, ser. no. 55201671, Germany), as BECs form the anatomical basis of the brains capillaries and its luminal surfaces are naturally exposed to circulating blood in vivo.

Chemical fixation Chemical fixation
Once bEnd5 monolayers reached confluence the inserts were removed using a lancet and were placed in protein and lipid cross-linking reagent such as 2.5% solution of glutaraldehyde (Fluka/ Sigma-Aldrich) was prepared in standard cell culture buffer-1X PBS solution with an osmolality akin to blood plasma within a range of 280-300 mOsmol/Kg and a pH 7.2 (Faso et al.,1994;Fischer et al., 2013). Following a 1h incubation period at room temperature (RT). The sample could be stored in a 2.5% glutaraldehyde fixative at 4°C overnight. Thereafter, the specimen was washed in 1X PBS (devoid of glutaraldehyde) for 2x5 minutes (min)  ' Following the dehydration of the fixed samples, it is required that the sample be dry before further processing could occur, this was performed using the Hitachi HCP-2 CPD. Evaporative drying of biological specimens could cause deformation and collapse from the native state of the sample. The deformities in the sample are often due to the surface tension of water, relative to evaporating air (Fischer et al., 2013). Therefore, CPD was performed by immersing biological samples in liquid with a lower surface tension to air (i.e. CO 2 ).
The dehydrated samples were transferred to and retained inside 10 mm diameter aluminium baskets. The basket holders were placed inside the CPD chamber and filled with liquid CO 2 . This step is critical, as the amount of liquid CO 2 injected into the chamber must be between 50% and 80% of the total chamber volume. The chamber would not reach the critical point (CP) with inadequate liquid CO 2 . The temperature was set to 20˚C for 15 min and then 38°C for 5min until the critical pressure was reached (73 kg/cm 2 ).

Bio-organic specimen coating for HR-SEM Bio-organic specimen coating for HR-SEM
It is imperative to ensure that the coating material does not compromise the surface details of the specimen during analysis. The coating's main function is to ensure sufficient surface conductivity, reduction of beam heating and radiation damage, as well as specimen volatility (Goldstein et al., 1992). Post CP drying, the BEC monolayers are coated with either a gold:palladium (Au:Pd) alloy by means of sputter-coating using a Quorum Q150T ES sputter coater, or with a thin layer of carbon during thermal evaporation of a carbon rod using an Emitech K950X carbon coater.
Sputter coating using gold:palladium Sputter coating using gold:palladium A Au:Pd alloy in an atomic ratio of 80:20 (Au:Pd 80:20) was used as the sputtered target material. The use of Au:Pd 80:20 is much en vogue compared to traditional gold only coatings. Palladium prevents Au agglomeration, which is known to produce large islands between 8 nm and 12 nm which result in an uneven coating, especially around the tallest structures in the specimen. This non-homogeneous coating invariably restricts resolution performance (Zhou et al., 2007). Conversely, Au:Pd 80:20 produces a finer, homogenous film with a particle grain size between 4 nm and 8 nm (Heu et al., 2019). This information is supported by a study that involved the imaging of blood capillaries using SEM by Zhou et al., (2007). In their study the luminal cell surface of a fenestrated adrenocortical endothelial cell after deposition of a 3 nm thick metal film of Au:Pd 60:40 is investigated, as by the high-resolution micrograph of Fig 2  Fig 2 ( Zhou et al., 2007).
For the purpose of this study, the thickness of the Au:Pd 80:20 coating is controlled by sticking to a standard sputtering time of 60 s. A chamber pressure of 10 -5 mbar is maintained beforehand, with a sputtering pressure of 10 -2 mbar used during coating. A sputtering current of 40 mA and tooling factor for Au:Pd of 2.3 are used with a quartz, crystal thickness monitor used to measure the thickness of the coating during sputtering. At the above experimental conditions, a deposition rate of roughly 5 nm/min is achievable at a sample-to-target distance of 50 mm. During deposition, the sample stage is rotated at a speed of 70 rotations per minute to ensure even coating across the specimen surface. Based on the above, a nominal coating thickness of 5 nm is thus deposited.
The deposition of layers of material (i.e. metal/non-metal) are denoted as films which range from nanometers to micrometers. Films can be classified into two types: (i) Physical Vapor Deposition (PVD) and (ii) Chemical Vapor Deposition (CVD) (Rath, 2014). To investigate the exact sample thickness, three different sputtering thicknesses are shown in Fig 3  Fig 3 below. From left to right, Au:Pd layers sputtered on soda-lime glass substrates for 30 s, 60 s and 120 s (s: seconds) are displayed. The reflectivity of the films decreases with increasing sputtering times, readily suggesting an increase in thickness of the films, with the sample deposited at 120 s exhibiting a very dark tinge.   (Zhou et al., 2007). Here the molecular detail on the luminal surface appears obscured, due to bulky coating, thus resulting in loss of ultrastructural detail.
To validate the sputtered thicknesses, surface profilometry was performed using a Veeco Dektak 6M Stylus Profilometer. A diamond stylus tip, with an average diameter of 12.5 µm, was scanned at a stepsize of 0.333 µm across the film for a total length of 3 mm. The thickness of the Au:Pd layer was determined by scanning the stylus across the film step, as indicated by the red arrows.
To validate the sputtered thicknesses, surface profilometry was performed using a Veeco Dektak 6M Stylus Profilometer. A diamond stylus tip, with an average diameter of 12.5 µm, was scanned at a stepsize of 0.333 µm across the film for a total length of 3 mm. The thickness of the Au:Pd layer was determined by scanning the stylus across the film step, as indicated by the arrows in  Fig 3,  Fig 3, a sputter time of 30s produces a film thickness of approximately 4 nm (shown by the red double arrows) which increases to roughly 5 nm after 60 s and 8 nm after 120 s. The spikes observed in the profilometry profiles are due to Au:Pd flakes attaching to the stylus tip. This is very common and indicative of the soft nature of the sputtered film surface. It must be noted that the above results on reasonably flat soda-lime glass produce even coatings, which is not always the case for specimens that are highly topographical. Previous results show that coatings of 30 s or less produce specimens that are unevenly coated and still experience surface charging during SEM analysis. Hence, to ensure evenly coated BEC monolayers and to avoid the previously mentioned challenges, a sputter coating time of 60 s (and thus Au:Pd 80:20 coating of 3-5 nm) is recommended for coating biological samples.

As shown in
Thermal evaporation of carbon Thermal evaporation of carbon In comparison, the BEC monolayers were coated with carbon during thermal evaporation. A 3 mm thick carbon rod is sharpened to a diameter (d) of 1.1 mm, with an evaporation length (l) totaling approximately 2 mm. The sharpened rod is mounted against a spring-loaded counter electrode in a vacuum, with the specimen placed a distance, r, from the carbon source. During evaporation, the chamber is pumped down to a vacuum of 10 -3 mbar, which increases to 10 -1 mbar for deposition. The current is then slowly increased to a max of 20 A and passed through the carbon rod, thereby heating it beyond the vaporization temperature of carbon; the vaporized carbon plumes subsequently coat the specimen.  ( 2) where ρ is the density of the source material. Fig 5  Fig 5 shows optical images of three carbon films deposited on soda-lime glass. From left to right, the films were deposited at a distance of 15 mm, 25 mm and 40 mm from the carbon source. As shown, the transparency of the films decreases with increasing distance, suggesting a decrease in film thickness. To confirm this, profilometry is once more employed and shown in   the carbon film integrity is of inferior quality than the Au:Pd films, which had high reflectivity indicative of a compact layer. A simple swab test also reveals that the carbon film is more powdered compared to the metal layers, as they delaminate far easier from the glass slides than the Au:Pd sputtered films. This is to be expected and can be explained by the relative chamber pressures (viz; the metal coatings were deposited in a cleaner chamber (10 -6 mbar) compared to the carbon coating at a pre-deposition vacuum of (10 -3 mbar)). Based on the above analysis, a standard sample distance of 25 mm was maintained for all specimens, implying a nominal carbon coating of 16 nm.  Optical images of carbon coatings deposited on soda-lime glass at a distance of 15, 25 and 40 mm from the carbon source with correlative profilometry measurements of carbon films deposited on soda-lime glass placed 15, 25 and 40 mm from the carbon rod. The glass level is indicated by the blue baseline, with the film step and thicknesses indicated by red arrows. The red and blue arrows correspond with each other, indicating the same place where the coating has taken place.